Honeys
Samples of acacia, buckwheat, and manuka honeys were obtained from Yamada Apiculture Center, Inc. (Tomata-Gun, Okayama, Japan).
Cell culture and reagents
All reagents were from Sigma-Aldrich, unless otherwise indicated. Human fibroblast cell line (46 BR.1N) was obtained from European Collection of Cell Cultures (ECACC). The cell line was originally derived from the skin of an individual with hypo-gammaglobulinemia, and immortalized by transfection with the plasmid pSV3neo, expressing SV40 T-antigen.[31] Cells were maintained at 37°C, 5% CO2, in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic mixture.
Calcein-acetoxymethylester (Calcein-AM) assay
The lipophilic, nonfluorescent Calcein-AM penetrates cell membranes and is then cleaved by intracellular esterases, yielding the hydrophilic fluorescent dye. Cells were settled overnight in 96-well plates (8,000 cells/well), incubated with honey for 24 h, washed with PBS, and then incubated for 30 min at 37°C with a solution of 2.5 μM calcein-AM in PBS. Plates were read in a fluorescence plate reader (Infinite 200 Pro, Tecan, Wien, Austria), by using 485-nm exc and 535-nm em filters.
Scratch wound assay
Fibroblasts were settled in 12-well plates and grown to confluence. Thereafter, scratch wounds were created in cell monolayers by using a sterile 0.1-10 μL pipette tip. After washing away suspended cells, cultures were refed with medium in the presence of different concentrations of honey for 24 h. Cells were then fixed in 3.7% formaldehyde in PBS for 30 min, and then stained with 0.1% toluidine blue at room temperature for 30 min. The width of the wound space was measured at wounding and at the end of treatments, using an inverted microscope equipped with a digital camera (Leica Microsystems). Digitized pictures of wounds were analyzed using the NIH Image J software. In a typical experiment, each group consisted of three different plates, i.e. a total of six wounds. Four measurements of wound width were made for each wound at randomly chosen points. Measurements were made by a single observer unaware of the treatments. Wound closure rates were determined as the difference between wound width at 0 and 24 h.
Cell migration assay
A cell migration assay was performed in transwell plates (8 μm pore size, Costar, Cambridge, MA). A total of 1×105 cells per well were seeded in the upper compartment of filters, while medium containing 0.1% honey was put in the lower compartment. After 24 h filters were removed and stained for 10 min with 0.5% crystal violet (145 mM NaCl, 0.5% formal saline, 50% ethanol), and then washed thrice with water. The upper side of filters was scraped using a cotton swab to remove cells that had attached but not migrated. Following PBS washing of filters, the dye was eluted from cells with 33% acetic acid, and measured at 540 nm in a plate reader (Infinite 200 Pro, Tecan, Wien, Austria). Chemotaxis was assessed by analyzing five independent filters.
Matrix metalloproteinase antibody array
Matrix metalloproteinases and their tissue inhibitors (MMP−1, −2, −3, −8, −9, −10, −13, and TIMP−1, −2, −3, −4) were determined in cell culture supernatants by an antibody array kit (RayBio MMP antibody array 1, RayBiotech, Norcross, GA) following the manufacturer’s protocol. The array consists of highly specific and well-characterized antibodies spotted on a nitrocellulose membrane. Cells were grown for 24 h in the presence of honey and conditioned media were then collected. Detection membranes were blocked with blocking buffer for 1 h at room temperature (RT) and then incubated with conditioned media. Membranes were washed, incubated with 1 ml of primary biotin-conjugated antibody at RT for 2 h, washed, incubated with 2 ml of horseradish peroxidase-conjugated streptavidin at RT, and then developed using enhanced-chemiluminescence solution (ECL), provided in the kit. Spots were observed, digitized and quantified with the ChemiDoc XRS system, using Quantity One Imaging software (Bio-Rad Laboratories, Hercules, CA).
Cytokine antibody array
Cytokines were quantified using the Human Cytokine Antibody Array kit 1.0 (Panomics, Inc., Redwood City, CA). The array (see above) allows for simultaneous detection of 18 cytokines and provides positive and negative controls. Cells were seeded in 12-well plates for 24 h, and then exposed to honey for 24 h. Collected conditioned media were then incubated for 1.5 h with membrane-immobilized capture antibodies specific to a particular cytokine protein. Unbound proteins were washed away. A second, biotin-conjugated antibody was allowed to bind for 1.5 h to a second epitope on the protein. Thereafter, 1 h incubation with streptavidin-horseradish peroxidase allowed visualization of proteins through detection of chemiluminescent signal. Spots were quantified as above.
Statistical analysis
Data were analyzed by ANOVA and the post hoc Tukey’s test, using the Instat software package (GraphPad Software, Inc, San Diego, CA). Median (EC50) and minimum (EC05) effective concentrations and their 95% confidence intervals were determined by using a downhill logistic dose-response curve developed by CSIRO, Australia:[32]
where T=top, S=Hill’s slope (negative for a downhill curve), D=honey concentration (% v/v). Statistical comparisons between EC50 values were based on overlapping or non-overlapping 95% CI.